Method for producing human anti-thymocyte immunoglobulins

ABSTRACT

Methods for producing improved anti-human thymocyte immunoglobulins from specific-pathogen-free animals are provided, without the need for an adsorption step on human tissues and the consequent drawbacks of such a step.

FIELD OF THE INVENTION

The present invention relates to a method for producing anti-humanthymocyte immunoglobulins.

BACKGROUND OF THE INVENTION

Anti-human thymocyte antibodies or immunoglobulins (Igs) are known to beselective immunosuppressants. They act on the immune response bydecreasing, by depletion, according to various mechanisms, the quantityof circulating lymphocytes of the blood and of the various lymphoidtissues, and probably by blocking or modulating their receptors. Theiruse is indicated in the context of organ transplants, for the preventionand treatment of transplant rejection, and also for the treatment ofacute graft versus host reaction. These immunoglobulins have also beenproposed in the treatment of medullary aplasia.

Preparations of this type of immunoglobulin were initially obtained byinjection of lymphatic cells into animals, rabbits or horses forexample, and then by extraction of the anti-thymocyte immunoglobulinsfrom the immune serum. However, the level of purity of thesepreparations was insufficient to render these immunoglobulins usable, inparticular in therapy (R. D Guttman, 1967, J. Exp. Med., 126:1099-1127).Antihuman erythrocyte or anti-basal membrane immunoglobulins were inparticular produced by the animal during the immunization. Severalhypotheses explain the appearance of these immunoglobulins: either thethymocyte suspensions contain traces of red blood cells (J. M. Rolland,1972, Pathology, 4(2): 85-122), or common surface antigens exist betweenT cells, B cells and erythrocytes, which at the current time is the mostplausible hypothesis.

Techniques were then developed in order to improve the purity of thepreparation. These techniques envisioned a step of hemadsorption, namelyadsorption on human red blood cells.

This step of hemadsorption makes it possible to remove the majority ofthe undesirable antibodies, such as the anti-erythrocyte antibodiespresent in the initial immune serum. A step of removal of theanti-tissue antibodies, on human tissues (such as placenta), was alsosometimes envisaged, in order to adsorb the anti-basal membraneantibodies in particular. Several anti-human thymocyte immunoglobulinsprepared according to these techniques are now available on the market,such as Thymoglobuline® (Imtix-Sangstat), Tecelac® (Biotest), or elseATGF® (Fresenius) or ATGAM® (Upjohn).

However, the methods described which comprise a step of adsorption onhuman tissues or red blood cells still exhibit drawbacks. In particular,in carrying out these methods, a 30% decrease in the level ofgammaglobulins contained in the serum, and therefore a not insignificantpart of the antibodies of interest, is observed. In addition, theremoval of the undesirable anti-erythrocyte antibodies is not complete.Now, these antibodies would be liable to cause the appearance ofhemolytic anemia in treated patients. Finally, the use of humanmaterial, such as erythrocytes, even purified and made safe, in themethod for isolating the anti-human thymocyte immunoglobulins, increasesthe potential risk of infection.

The authors of the present invention have therefore sought to develop animproved method for producing anti-human thymocyte immunoglobulins.

SUMMARY OF THE INVENTION

The present invention relates to a method with no step of adsorption onhuman biological material, for producing a preparation of anti-thymocyteimmunoglobulins which is sufficiently pure for use in therapy inparticular.

More precisely, a subject of the invention is a method for producinganti-purified human thymocyte immunoglobulins, which comprises the stepsconsisting in:

-   -   (a) injecting a cellular preparation of human thymocytes into a        specific-pathogen-free (SPF) animal;    -   (b) collecting the immune serum produced by the animal;    -   (c) isolating the anti-human thymocyte immunoglobulins from the        serum, with no adsorption on human biological material.

More particularly, the expression “immunoglobulins from serum” isintended to mean the total fraction of gammaglobulins from the serumafter immunization.

The expression “adsorption on human biological material” is intended tomean in particular hemadsorption on red blood cells or adsorption onhuman tissues (such as placenta in particular), stroma or crude extractsof these tissues.

DETAILED DESCRIPTION OF THE INVENTION

The SPF animals are breeding animals whose environment and food arestrictly controlled according to health standards. Reference is alsomade to animals “with controlled health status”. The SPF animals arebred in closed, sterilized compartments, the air environment beingfiltered, for example through a HEPA filter (sterilizing filtration),and the water and foodstuff being decontaminated before introduction,which makes it possible to eliminate any pathogenic element from theirdirect environment (Yanabe et al. Exp. Animal 48(2), 101-106 (1999)).The term “pathogen” here denotes an infectious agent capable of causinga clinical disease and/or of modifying the biological response of theanimal with regard to the desired use. The SPF status refers to a list(which evolves) of microorganisms and of methods of control (clinical,serological, histological, or using culturing) for detecting thetargeted microorganisms or, conversely, demonstrating their absence.

Use is preferentially made of horses or goats, more preferably rabbits,for the production of immune serum. In fact, only IgG1s are present inrabbit serum, which facilitates the process of antibody purification.Rabbit IgG also shows a high affinity for the human Fc receptor,allowing the development of powerful cytotoxic antibodies directedagainst human T cells.

The cellular preparation of human thymuses can be obtained either fromcells of lines in culture, or from fresh thymocytes which are purifiedpreferentially from human thymus fragments or optionally from, forexample, suspensions of spleen, tonsils, lymph nodes, thoracic tracheaor peripheral blood. The human thymus fragments can in particular beeasily removed during surgical acts, in particular subsequent to cardiacsurgery on children. Virological tests are carried out on the donor'sblood in order to avoid any contamination and to eliminate anycontaminated thymic fragment showing a positive serological result.

The injecting of the human thymocytes and the collecting of the immuneserum from the SPF animal are carried out according to standardtechniques of those skilled in the art.

Isolation of the anti-human thymocyte immunoglobulins from the serummakes it possible to eliminate the undesirable proteins. It can becarried out using, for example, ion exchange chromatography, preferablyon a column, and/or one or more precipitations.

Advantageously, such a precipitation can be carried out in twosuccessive steps using an immunoglobulin precipitating reagent. Thereagent preferentially used is sodium sulfate.

The chromatography step enables retention of the loaded impurities by anion exchange resin such as anions (DEAF). As regards the IgGs, they arenot retained by the column and are rapidly removed from the column,which makes it possible to harvest them selectively. Chromatography on aspecific affinity column, which removes the remaining undesirableantibodies, is also advantageous.

A subject of the invention is also the isolated antihuman thymocyteimmunoglobulins which can be prepared using the method of the invention.

These immunoglobulins can also be designated “antihuman lymphocyteimmunoglobulins” since they recognize human lymphocytes when they arebrought into contact with these lymphocytes, for example when injectedinto patients for the purpose of immunodepletion.

Moreover, the present invention is also directed toward the use of theseimmunoglobulins for producing a medicinal product intended to decreasethe quantity of circulating lymphocytes of the blood and of the lymphoidtissues in a human patient.

A subject of the invention is also a method of therapeutic treatment inwhich a therapeutically effective quantity of immunoglobulins thusobtained is administered to a patient requiring his or her quantity oflymphocytes to be decreased. These immunoglobulins are particularlyuseful in the context of organ transplants.

The anti-human thymocyte immunoglobulins prepared according to themethod described above exhibit a greater specific activity than theanti-thymocyte immunoglobulins prepared using a method including ahemadsorption step, as emerges in Example 2.1 presented below.

Deleting the step of adsorption on human biological material makes itpossible to produce immunoglobulins without any contact with a humanderivative. This leads to the elimination of any possible viralcontamination or contamination with unconventional agents of the priontype. In addition, any contamination associated with hemadsorption,namely contamination with the hemoglobin released by the human red bloodcells during the hemolysis engendered by this step, is avoided.

The method of the invention makes it possible to obtain a preparation ofanti-thymocyte immunoglobulins in which the number of immunoglobulinscapable of cross reacting with other blood cells (erythrocytes,neutrophils, etc.) is considerably reduced. In addition, the fact thathemadsorption is not used in preparing the anti-thymocyte antibodiesmakes it possible not only to improve the safety aspect, but alsoreduces the length and the cost of the preparation method. In fact,hemadsorption conventionally uses whole human red blood cells which arefresh and formalin-treated, and requires large quantities of cells to betreated, making the production of antithymocyte immunoglobulinsrelatively restrictive in industrial terms.

The following examples illustrate the invention without limiting thescope thereof.

EXAMPLES Example 1 Production of Immunoglobulins

1.1 Purification of Thymocytes

Human thymus fragments are, after removal and grinding, filtered andplaced in suspension.

The cell suspension is then filtered through a nylon cloth and subjectedto centrifugation at 1800 rpm at 5° C.

20 ml of Ficoll are added to 10 ml of cell solution containing 2 to7×10⁹ cells and the mixture is centrifuged at 2000 rpm at 5° C. Twosubsequent centrifugations are carried out at 1800 rpm. The cellularpreparations are then stored at 5° C. overnight and are then dilutedbefore being injected into SPF rabbits. The thymocytes can also beconserved by freezing.

1.2 Isolation of Antibodies

The rabbit immune serum is collected in the course of several bleedsbetween D20 and D30, and can be frozen for storage. During thepreparation, batches of serum are formed which are gradually returned toambient temperature.

These batches of rabbit serum are decomplemented in order to remove thecomplement proteins, by bringing them to a temperature of 56□C±2□C for aperiod of 30 to 45 min.

The rabbit serum is purified for the immunglobulin fraction bychromatography on an anion exchange resin (DEAE) at ambient temperaturefollowed by two precipitations with sodium sulfate.

After a further step of filtration, concentration and diafiltration, theanti-thymocyte immunoglobulins are pasteurized at a temperature of 60°C. for 10 hours in order to ensure the viral safety thereof.

According to one variant, the steps of filtration, concentration,diafiltration and precipitation of the immunoglobulin fraction (forexample alcoholic fractionation of the COHN type or ammonium sulfatefractionation) can be carried out before the chromatography step.

The solution of immunoglobulins can, after formulation and sterilizationby filtration, be conserved in the liquid state in a solution of 5 to 25mg/ml or, according to one variant, in the lyophilized state. A seriesof quality controls is executed, including physicochemical tests, safetytests (presence of pyrogenic agents, of antiplatelet activity),sterility and purity tests and also lymphocytotoxic activity tests(inhibition of rosette formation in vitro and allogenic skin graftsurvival in monkeys).

Example 2 Comparative Analysis of the Effectiveness and the Innocuity ofthe Immunoglobulins Obtained Using the Method of the Invention, withRespect to Conventional Immunoglobulins (Obtained with Hemadsorption)

3.1 Measurement of the Specific Activity In Vitro

Two batches of immunoglobulins were used for the comparative study ofthe specific activity: a standard anti-thymocyte immunoglobulin (ATG)batch and a nonhemadsorbed ATG batch.

To determine the specific activity of the ATGs, i.e. to evaluate thequantity of immunoglobulins capable of binding to human lymphocytes, atechnique of flow cytometry coupled to indirect immunofluorescence isused. Whole monkey blood (containing all blood cells) is incubated withthe ATG batches at various concentrations. The excess of unattachedimmunoglobulins is rinsed off and a second antibody, labeled withfluorescein isothiocyanate (FITC), is added so as to attach to the boundATGs. The amount of binding of the ATGs to the surface of the cells isthen revealed after differentiation of the cells as a function of theirsize using flow cytometry.

The results show a greater specific activity of the non-hemadsorbedATGs. Thus, to obtain the same binding to lymphocytes, 2.73 μg/ml ofnon-hemadsorbed ATG are necessary, where 7.23 μg/ml of standard ATG arenecessary, i.e. 2.65 times more. The hemadsorption step thereforedecreases the binding capacity and the specific activity of the ATGs.

3.2 Measurement of the Activity of the ATGs in Vivo in the Skin GraftTest in Monkeys

Three groups of monkeys were selected for carrying out this test: acontrol group (n=5), a group receiving 5 mg/kg of standard ATG (n=8) anda group receiving 5 mg/kg of ATG not hemadsorbed (n=3) initially. A50/50 dilution of the non-hemadsorbed ATGs is carried out in order toobtain a specific activity equivalent to that of the standard batch.

The skin graft test consists in performing, on DO, four allografts(originating from an animal in which at least two of the HLA antigenicdeterminants are different) and an autograft of skin on the back of eachanimal. The doses of ATG are then administered in vivo for three weeksfrom D-1.

The results show that the mean survival times of the allografts areequivalent for the two groups of treated monkeys: 23 days (±4.16) forthe standard ATG group and 21.3 days (±7.3) for the non-hemadsorbed ATGgroup. They are also greater than the control: 9.4 days (±1.5).

The prolonging of the survival time of the skin graft correlates withthe lymphocyte depletion induced by the administration of ATG. Thelymphopenia follows an evolution which is comparable between the twobatches: maximum decrease in the level of lymphocytes at D5, thengradual return to normal values around D20.

Consequently, the diluted batch of non-hemadsorbed ATGs is equivalent,in terms of effective in vivo in the primate, to the undiluted standardATG batch.

3.3 Evaluation of the Risks of Hemolytic Anemia Subsequent toAdministration of ATGs

In the context of the same study, blood tests are carried out regularlyuntil the end of the test and show that the evolution of the hemoglobinlevel is identical in the two groups of treated monkeys. A decrease inthis level is observed up to D9, until this level gradually returns tonormal values. Two phenomena are responsible for this evolution:firstly, the anemia is of the iatrogenic type, subsequent to therepeated taking of blood samples. This anemia is identical in the threegroups of monkeys. Secondly, an effect specific to the ATGs causes asubstantial decrease in the hemoglobin level from D1 in the treatedgroups, this decrease being identical with the standard ATG or with thenon-hemadsorbed ATG.

In addition, in order to detect a possible hemolytic cause for theanemia, the levels of haptoglobin and of oromucoid are also measured. Inthe event of hemolysis, haptoglobin becomes unmeasurable. Now, these twolevels increase from D4 for the two groups, this increase reflecting aninflammatory activity.

Thus, the hematologic safety evaluated in the animals treated with thenon-hemadsorbed ATG is as satisfactory as in the animals treated withthe hemadsorbed ATG.

1. A method for producing a therapeutic preparation of anti-humanthymocyte immunoglobulins for use in human therapy, comprising the stepsof: a. injecting a cellular preparation of human thymocytes obtainedfrom human thymus into a specific-pathogen-free (SPF) animal; b.collecting the immune serum produced by the animal; and c. isolating theanti-human thymocyte immunoglobulins from the collected immune serum;wherein said method includes no step of adsorption on human biologicalmaterial in order to produce said therapeutic preparation of anti-humanthymocyte immunoglobulins.
 2. The method according to claim 1, whereinthe SPF animal is a rabbit.
 3. The method according to claim 1, whereinthe isolation step (c) comprises a chromatography step.
 4. The methodaccording to claim 3, wherein the chromatography step compriseschromatography on an ion exchange resin on a column.
 5. The methodaccording to claim 4, wherein said ion exchange resin is an anionexchange resin.
 6. The method according to claim 5, wherein said anionexchange resin is DEAE.
 7. The method according to claim 3, wherein thechromatography step further comprises chromatography on a specificaffinity column.
 8. The method according to claim 3, wherein saidisolation step (c) further comprises at least one precipitation step. 9.The method according to claim 8, wherein said at least one precipitationstep uses an immunoglobulin-precipitating reagent.
 10. The methodaccording to claim 9, wherein said immunoglobulin-precipitating reagentis sodium sulfate.
 11. The method according to claim 1, wherein thecellular preparation of human thymocytes obtained from human thymus usedin step (a) comprises fresh human thymocytes.